Abstract B109: Identification of small molecule activators targeting the JH2 pseudokinase domain of TYK2

Abstract Background: Cancer immunotherapy has emerged as a promising strategy to eliminate cancer cells by harnessing the body's immune system, yet there is a high demand for new approaches to enhance its effectiveness. TYK2 plays a crucial role in immune responses, and recent studies have shown that TYK2 signaling is essential for anticancer immunity. Therefore, activating TYK2 signaling could be a novel therapeutic strategy to enhance tumor immunosurveillance. Here, we report the design and synthesis of aminopyridine analogs that can activate TYK2 by binding to its JH2 pseudokinase domain. Materials and methods: A focused library of aminopyridine analogs, derived from a known JH2 binder, was constructed using copper-catalyzed azide-alkyne cycloaddition. The activation potential of compounds was evaluated in an enzymatic assay by measuring substrate phosphorylation using a recombinant human TYK2 protein containing both JH1 and JH2 (JH1/JH2 TYK2). Compound-induced activation was expressed as fold-change (FC) relative to the control. The X-ray co-crystal structure of selected compounds with the TYK2 JH2 domain was determined to discuss the potential mechanism of action. Results: We designed and synthesized 16 aminopyridine analogs with various substituents, and screened their activation potency using JH1/JH2 TYK2. Among the 16 aminopyridine analogs, the compound bearing an azide group increased TYK2 activity (FC = 2.1 at 1 μM). To validate this hit compound, the azide group was replaced with small aliphatic groups. The acetylene analog exhibited similar potency, suggesting that linear-shaped substituents such as azide or alkyne may be key steric elements influencing TYK2 activation. Subsequently, we derivatized the acetylene analog to introduce various substituents at the acetylene head position. Notably, HTO-066 exhibited strong activation potency with a maximum FC of 25.9. We solved the X-ray co-crystal structure of HTO-066 bound to the TYK2 JH2 domain and found that HTO-066 induced a conformational change in the JH2 domain compared with the inhibitor-bound structure. Conclusions: We have discovered a novel alkyne analog, HTO-066, that activates TYK2 enzymatic activity, presumably by destabilizing the JH2-JH1 autoinhibitory complex. Co-crystal structural analysis suggested that HTO-066 induces a conformational change in the JH2 domain, which may interfere with the formation of the JH2-JH1 autoinhibitory complex. Conflict of interest: Board of Directors: M. Sawa is Director & Chief Scientific Officer of Carna Biosciences, Inc. Other Substantive Relationships: T.Kinoshita receives research funding from Carna Biosciences, Inc. Citation Format: Hirokazu Matsumoto, Tien-Cheng Wang, Aoi Matsushita, Haruka Taniguchi, Yu Nishioka, Mariko Hatakeyama, Takayoshi Kinoshita, Masaaki Sawa. Identification of small molecule activators targeting the JH2 pseudokinase domain of TYK2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B109.